Methods, cells and reagents for production of isoprene, derivatives and intermediates thereof

Abstract

This application describes methods, including non-naturally occurring methods, for biosynthesizing 3-hydroxy-3-methylglutaryl-coA and intermediates thereof, as well as non-naturally occurring hosts for producing 3-hydroxy-3-methylglutaryl-coA. This application also describes methods, including non-naturally occurring methods, for biosynthesizing isoprene and intermediates thereof, as well as non-naturally occurring hosts for producing isoprene.

1 .- 10 . (canceled) 
     
     
         11 . A non-naturally occurring host capable of producing 3-hydroxy-3-methylglutaryl-CoA, said host comprising:
 at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 1.3.8.4 enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 6.4.1.4 enzyme; and   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 4.2.1.18 enzyme;   and wherein said host further comprises:   (a) at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 1.2.7.7 or EC 1.2.1.-enzyme;   (b) at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 4.1.1.74 or EC 41.2.1.43 enzyme,
 at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 1.2.1.39 or EC 1.2.1.5 enzyme, and 
 at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 6.2.1.2. enzyme; or 
   (c) at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 1.2.7.7 or EC 1.2.1.-enzyme,
 at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 4.1.1.74 or EC 41.2.1.43 enzyme, 
 at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 1.2.1.39 or EC 1.2.1.5 enzyme, and 
 at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 6.2.1.2. enzyme. 
   
     
     
         12 .- 13 . (canceled) 
     
     
         14 . The host of  claim 11 , wherein said host is capable of producing isoprene and comprises:
 at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 1.1.1.34 enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 2.7.1.36 enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 2.7.4.2 enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 4.1.1.33 enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 5.3.3.2 enzyme; and   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 4.2.3.27 enzyme.   
     
     
         15 . The host of  claim 11 , wherein said host is capable of producing 3-hydroxy-3-methylglutaryl-CoA, said host further comprising at least one of:
 at least one endogenous enzyme capable of enzymatically converting 4-methyl-2-oxopentanoate to 3-methylbutanoyl-CoA;   at least one endogenous enzyme capable of enzymatically converting 4-methyl-2-oxopentanoate to 3-methylbutanal;   at least one endogenous enzyme capable of enzymatically converting 3-methylbutanal to 3-methylbutanoate;   at least one endogenous enzyme capable of enzymatically converting 3-methylbutanoate to 3-methylbutanoyl-CoA;   at least one endogenous enzyme capable of enzymatically converting 3-methylbutanoyl-CoA to 3-methylbut-2-enoyl-CoA;   at least one endogenous enzyme capable of 3-methylbut-2-enoyl-CoA to 3-methyl-glutaconyl-CoA; and   at least one endogenous enzyme capable of 3-methyl-glutaconyl-CoA to 3-hydroxy-3-methylglutaryl-CoA.   
     
     
         16 . The host of  claim 11 , wherein at least one of the exogenous nucleic acids is contained within a plasmid. 
     
     
         17 . The host of  claim 11 , wherein at least one of the exogenous nucleic acids is integrated into a chromosome of the host. 
     
     
         18 .- 19 . (canceled) 
     
     
         20 . The host of  claim 11 , wherein the host is a prokaryotic host. 
     
     
         21 . The host of  claim 20 , wherein the host is a prokaryotic host from the genus  Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Bacillus , or  Rhodococcus.    
     
     
         22 . The host of  claim 21 , wherein the host is from the genus  Cupriavidus.    
     
     
         23 . The host of  claim 22 , wherein the host is  Cupriavidus necator.    
     
     
         24 . The host of  claim 11 , wherein the host is a eukaryotic host. 
     
     
         25 . The host of  claim 24 , wherein the host is a eukaryotic host from the genus  Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula , or  Kluyveromyces.    
     
     
         26 . (canceled) 
     
     
         27 . The host of  claim 11 , wherein the host is capable of endogenously producing isoprene via a non-mevalonate pathway. 
     
     
         28 .- 42 . (canceled) 
     
     
         43 . The host of  claim 11 , wherein said host comprises:
 at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 1.2.7.7 or EC 1.2.1.-enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 1.3.8.4 enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 6.4.1.4 enzyme; and   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 4.2.1.18 enzyme.   
     
     
         44 . The host of  claim 11 , wherein said host comprises:
 at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 4.1.1.74 or EC 4.1.1.43 enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 1.2.1.39 or EC 1.2.1.5 enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 6.2.1.2. enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 1.3.8.4 enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 6.4.1.4 enzyme; and   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 4.2.1.18 enzyme.   
     
     
         45 . The host of  claim 11 , wherein said host comprises:
 at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 1.2.7.7 or EC 1.2.1.-enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 4.1.1.74 or EC 4.1.1.43 enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 1.2.1.39 or EC 1.2.1.5 enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 6.2.1.2. enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 1.3.8.4 enzyme;   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 6.4.1.4 enzyme; and   at least one exogenous nucleic acid encoding a polypeptide having the activity of an EC 4.2.1.18 enzyme.